Over the last decade, it has become apparent that dysregulation of the immune system is related to several serious diseases, including include diabetes, cardiovascular diseases, and chronic inflammation. Millions of Americans are affected by these diseases and results in increased healthcare costs. The imbalance of the immune/inflammatory responses in these diseases ultimately leads to cellular damage and pathophysiologic dysfunction like neuropathy and ischemia. While clinicians are working to identify key biomarkers to use in determining or monitoring the seriousness of these diseases, little has been done to identify the direct effects that inflammatory biomarkers have on the common target of the microvasculature. The release of acute phase proteins (APP), including haptoglobin, serum amyloid A, and C-reactive protein, occurs in many of these diseases, yet the underlying cellular impact of these inflammatory biomarkers is not well understood. Our hypothesis is that inflammatory mediators, such as APPs, act directly on the endothelial cells of the microvasculature, activating signaling pathways and cellular mechanisms that alter the normal endothelial barrier function. This proposal focuses on examining changes in the microvasculature following exposure to APPs to gain a better understanding of the underlying mechanisms that lead to plague development and disrupted endothelial transport. Using an in vitro BBB model (primary isolated rat brain microvessel endothelial cells, RBMEC) and a peripheral microvascular endothelial model (human umbilical vein endothelial cell line, HUVEC) we will examine the cellular mechanisms that are likely involved in structural and molecular changes of the microvascular endothelial barrier in response to APPs. Functional (BBB permeability), structural (expression of tight junction proteins) and molecular (release of related inflammatory cytokines) assessments will be carried out to establish a link between clinical findings of elevated levels of C-reactive protein, haptoglobin, etc. and the pathophysiological changes observed in the microvasculature of patients from afore mentioned diseases. The results from this study will provide the proof of concept for further investigation into clarifying cellular signaling pathways that are activated by these inflammatory biomarkers as well as identify ideal pharmacological treatments that will reduce the risks and improve the quality of life in patients with these diseases. The focus of this research proposal is to enhance our understanding of cellular mechanisms in the microvascular endothelium which respond to inflammatory mediators, especially acute phase proteins. The results of this study will provide invaluable information regarding the effects that acute phase proteins (i.e., C-reactive protein, haptoglobin, and serum amyloid A) stimulate in the microvascular endothelial barrier including permeability and related structural proteins that regulate these barriers. PUBLIC HEALTH RELEVANCE: The clinical impact of these studies will provide targets for therapeutic treatment as well as preventive measures for those patients with cardiovascular and inflammatory-related diseases. [unreadable] [unreadable] [unreadable]